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. 2015 Mar 19;2(2):e80.
doi: 10.1212/NXI.0000000000000080. eCollection 2015 Apr.

P2Y12 expression and function in alternatively activated human microglia

Craig S Moore  1 , Ariel R Ase  1 , Angham Kinsara  1 , Vijayaraghava T S Rao  1 , Mackenzie Michell-Robinson  1 , Soo Yuen Leong  1 , Oleg Butovsky  1 , Samuel K Ludwin  1 , Philippe Séguéla  1 , Amit Bar-Or  1 , Jack P Antel  1
Affiliations

P2Y12 expression and function in alternatively activated human microglia

Craig S Moore et al. Neurol Neuroimmunol Neuroinflamm. .

Erratum in

Abstract

Objective: To investigate and measure the functional significance of altered P2Y12 expression in the context of human microglia activation.

Methods: We performed in vitro and in situ experiments to measure how P2Y12 expression can influence disease-relevant functional properties of classically activated (M1) and alternatively activated (M2) human microglia in the inflamed brain.

Results: We demonstrated that compared to resting and classically activated (M1) human microglia, P2Y12 expression is increased under alternatively activated (M2) conditions. In response to ADP, the endogenous ligand of P2Y12, M2 microglia have increased ligand-mediated calcium responses, which are blocked by selective P2Y12 antagonism. P2Y12 antagonism was also shown to decrease migratory and inflammatory responses in human microglia upon exposure to nucleotides that are released during CNS injury; no effects were observed in human monocytes or macrophages. In situ experiments confirm that P2Y12 is selectively expressed on human microglia and elevated under neuropathologic conditions that promote Th2 responses, such as parasitic CNS infection.

Conclusion: These findings provide insight into the roles of M2 microglia in the context of neuroinflammation and suggest a mechanism to selectively target a functionally unique population of myeloid cells in the CNS.

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Figures

Figure 1
Figure 1. P2Y12 expression is significantly elevated in human microglia compared to monocyte-derived macrophages
(A) Using TaqMan quantitative real-time PCR, P2Y12 expression was measured in RNA isolated from resting human monocyte-derived macrophages and microglia (both fetal and adult). Compared to monocyte-derived macrophages, delta cycle threshold (CT) values were much lower in both human fetal and adult microglia populations, which is indicative of higher P2Y12 expression. Error bars represent mean ± SEM; n = 4 samples/condition; ****p < 0.0001. (B) Using immunocytochemistry, resting CD68+ human microglia expressed P2Y12; no immunoreactivity was observed in resting macrophages.
Figure 2
Figure 2. P2Y12 expression is upregulated in M2-polarized human microglia
(A, B) Using TaqMan quantitative real-time PCR, P2Y12 expression was measured in M1- and M2-polarized human microglia (both adult and fetal). Compared to unpolarized (Unpol.) and M1-polarized cells, M2 microglia had significantly elevated P2Y12 expression. Error bars represent mean ± SEM; n = 5/condition for microglia, n = 3/condition for macrophages; *p < 0.05, ***p < 0.001 compared to unpolarized. (C) Using flow cytometry, gating on the live human microglia population, mean fluorescence intensity of P2Y12 was increased in M2-polarized (red line) adult microglia (D) and fetal microglia (E) compared to resting conditions (blue line). Shaded histogram represents isotype control.
Figure 3
Figure 3. ADP-evoked intracellular calcium responses discriminate between human microglia and macrophages while increased ADP-evoked intracellular calcium responses are observed in M2-polarized human microglia
(A) Averaged traces of intracellular calcium ([Ca2+]i) levels following a short application of ADP in fetal human microglia. The P2Y12 selective antagonist PSB0739 strongly blocks ADP-induced [Ca2+]i. (B) Quantification of ADP-evoked [Ca2+]i responses in the absence or presence of PSB0739. (C) ADP-evoked [Ca2+]i is minimal in human macrophages; however, the cells respond strongly to ATP. (D) Quantification of ADP- and ATP-induced [Ca2+]i transients. (E) Averaged traces of [Ca2+]i levels following application of ADP in unpolarized human fetal microglia and (F) comparison with responses to ADP in M2-polarized cells. (G) ADP-evoked [Ca2+]i responses in both unpolarized (Unpol.) and M2-polarized cells are blocked by the P2Y12 antagonist PSB0739, summary quantification. Error bars represent mean ± SEM; n = 14–35 cells/condition; ***p < 0.001.
Figure 4
Figure 4. ADP-induced migration of human microglia is blocked by a P2Y12 antagonist
(A) Using 10 μm cell migration chambers followed by crystal violet staining, ADP induced a significant migration of human microglia, whereas a 30-minute preexposure of a P2Y12 antagonist (PSB0739; 10 μM) significantly decreased ADP-induced migration after 6 hours. Results were quantified in both adult (B) and fetal (C) microglia. (D–F) Using silicon migration chambers, preexposure of a P2Y12 antagonist also significantly reduced migratory distance of human microglia. For panels B and C, error bars represent mean ± SEM; n = 4/condition for microglia. For panel F, shaded box represents 95% confidence interval; error bars represent minimum and maximum values. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 5
Figure 5. ADP-induced inflammatory responses require P2Y12 receptors in human microglia
Increasing concentrations of ADP (5, 50, and 200 μM) increased tumor necrosis factor (TNF) and interleukin (IL)-6 release in human adult microglia (A, C) and fetal microglia (B, D), with maximal response reached at 200 μM after 6 hours. A 1-hour pretreatment with a P2Y12 receptor antagonist (PSB0739) dose-dependently decreased TNF and IL-6 cytokine expression induced by ADP (200 μM) as measured by ELISAs. Error bars represent mean ± SEM; n = 4/condition; **p < 0.01 compared to ADP (200 μM) treatment alone.
Figure 6
Figure 6. P2Y12 is highly expressed in CD68+ and CD163+ cells during parasitic brain infection
In a case of Schistosoma mekongi infection, hematoxylin and eosin–stained sections show evidence of the brain parenchyma containing granulomas and helminthic ova surrounded by inflammatory cells (A). Within the granulomas, immunohistochemistry experiments showed the presence of several CD68+ (non-myeloid cell-specific) cells (B) and CD163+ (M2 marker) (C) cells that positively stained for P2Y12. White arrows in the higher magnified panel point toward double-positive CD163+/P2Y12+ cells.
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